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Abstract 2019-2020: Comparative nuclear and mitochondrial genomics of animal and human Trypanosoma brucei

Background
The Western and Central African form of Human African trypanosomiasis (HAT), also known as sleeping sickness, is a vector-borne disease caused by Trypanosoma brucei gambiense and transmitted by tsetse flies. The World Health Organization (WHO) targeted T.b. gambiense HAT (gHAT) for elimination as a public health problem and elimination of
transmisson. Within this context, new highly sensitive molecular diagnostic tools are required to investigate the role of undiagnosed patients, asymptomatic carriers and
potential animal reservoirs in sustaining HAT transmission and re-emergence. To reliably demonstrate T.b. gambiense infections, we want to identify a new molecular marker that
is fully specific for T.b. gambiense (type I) from diverse geographical origins and that is more sensitive than current genetic markers. To this end, we decided to investigate the
mitochondrial minicircles as potential candidate markers as they occur in high copy numbers .

Methods
DNA of 38 Trypanosoma brucei strains, from 13 different countries and isolated from man and animals between 1961 and 2009, was extracted by the phenol-chloroform method and
sequenced by the DNA nanoball sequencing technology (DNBSEQTM, BGI, China). Reads were aligned against the reference genome TbTREU927 v5.1 available on TriTrypDB using SMALT v0.7.6. Per site sequencing coverage was computed with SAMtools v1.9 and used to calculate average read depths in non-overlapping 10kb genomic windows in python. Single Nucleotide Polymorphisms (SNP) were identified using GATK v4.1.4 HaplotypeCaller and filtered with BCFtools v1.10.2. Genome-wide SNP distribution and SNP loci variation within subspecies were calculated and visualized in R v3.6.3. Phylogenetic network analysis was done in SplitsTree5 and Principle Component Analysis in ADEGENET v2.1.3 as implemented in R.
Unmapped reads were quality trimmed using fastp v0.20.0. Mitochondrial minicircle contigs were assembled with a kmer list of 99, 109, 119, circularized (removing overlapping contig ends) and polished (reorientation and alignment) using KOMICS v.1.8. Following alignment of unmapped reads to each minicircle contig, we assessed the quality of the minicircle assembly by calculating the number of mapped reads, properly paired reads and the proportion of properly paired reads containing the Conserved Sequence Block 3 (CSB3) (i.e. a highly conserved 12-mer within the minicircle). The resulting minicircle contigs of all strains were clustered into minicircle sequence classes (MSC) based on a minimum percent identity using VSEARCH v2.14.2. Maxicircles were assembled by alignment to the T. brucei maxicircle reference M94286. SNP-calling and phylogenetic analysis were performed as previously described.

Results
Phylogenetic network analyses using a total of 315.989 genome-wide SNPs and 194 SNPs within the mitochondrial maxicircle revealed the presence of four clusters that
corresponded to the four known T. brucei subspecies: T.b. brucei (n=7), T.b. gambiense (n=18), T.b. gambiense type II (n=3) and T.b. rhodesiense (n=4). Six strains showed an
uncertain/hybrid ancestry. T.b. gambiense forms a monophyletic group of parasites showing a low level of intra-group diversity compared to other subspecies (5- to 10-fold
lower number of SNPs). A total of 9.110 minicircle contigs were assembled across all strains, of which 78% were successfully circularized. Clustering analyses retrieved 3.053
minicircle sequence classes (MSC) at a percent identity of 97%. Six MCS’s were found to be T.b. gambiense specific (i.e. occur in all 18 T.b. gambiense strains).

Conclusion
We were able to identify six promising candidates for new T.b. gambiense specific diagnostic markers and this based on strains from diverse geographical origin: Burkina
Faso (n=1), Cameroon (n=2), Congo-Brazzaville (n=2), DRC (n=10), Côte d’Ivoire (n=2) and Sudan (n=1).